We have invented a new photometric immunoassay method for detecting and quantifying various analytes in physiological fluids. Our invention is directed in particular to an initial rate photometric immunoassay method.
There is a continuing need for a rapid, accurate, and reproducible method for the detection and quantification of various naturally synthesized substances, such as antibodies, antigens, and hormones at low concentrations. Additionally, there is an extensive and pervasive need to detect other biologically active substances such as drugs and toxins in physiological fluids. Detection of therapeutic drugs and drugs of abuse can be very important to, for example, medical therapy, law enforcement and employment decisions.
Clinical chemistry laboratory tests are an important part of a health care system. Physicians frequently use such tests to monitor serum protein and therapeutic drug levels where only a narrow safe serum level for a drug may exist. Typically, microgram concentrations of a particular drug in a physiological fluid, such as serum, plasma, urine, amniotic, pleural or cerebrospinal fluid must be determined to permit effective medical treatment or to detect nontherapeutic use of opiates such as heroin, morphine and cocaine. With billions of clinical chemistry tests being performed annually, the speed, accuracy and cost control of such tests are important objectives.
A number of commonly used clinical chemistry tests detect and quantify various substances present in physiological fluids through an immunoprecipitation reaction. An immunoprecipitation reaction can occur when two reaction partners, each with a specific binding affinity for the other, are combined in a suitable liquid medium. The reaction partners can be an antigen and a specific binding partner for the antigen, such as an antibody. Generally, one of the reaction partners is present in an unknown amount in a sample of the physiological fluid, and is called the analyte. Typically, the liquid medium is a buffered aqueous solution. Once initiated, the immunoprecipitation reaction results in the formation of immunoprecipitates, or antibody-antigen complexes that are usually insoluble, but which can also be soluble, in the liquid medium.
The presence of immunoprecipitates in the liquid medium can change optical properties, such as light scattering and light absorption properties of the liquid medium, by attenuation of incident light energy. These changes can be detected by an appropriate photometer. The photometer can be calibrated to permit detection and quantification of an analyte. Calibration is typically carried out by conducting the immunoprecipitation reaction with known amounts of an analyte of interest, to derive a standard or calibration curve. The calibration curve can show a level of light attenuation, for example light absorption, by the liquid medium versus amount of analyte present per unit volume of a physiological fluid.
Photometric immunoassay techniques include nephelometry and turbidimetry. In nephelometric immunoassay, a photometer is used to measure the reflection or scatter of light by immunoprecipitates towards a light detector. The immunoprecipitates can be aggregates of an analyte and a specific binding partner for the analyte, or aggregates of an analyte-conjugate and the specific binding partner. The amount of light scattered by the immunoprecipitates is directly proportional to the number of immunoprecipitates present, which typically increases as the immunoprecipitation reaction proceeds. This proportionality permits a quantitative determination of analyte concentration. In turbidimetric immunoassay, an attenuation or reduction of light energy passing through a liquid medium containing immunoprecipitates is measured by a light detector placed in the light path. The light energy reduction can be caused by reflection, scatter, and absorption of the incident light by the immunoprecipitates. The amount of light reduction caused by the immunoprecipitates is, again, directly proportional to the number of immunoprecipitates present, permitting a quantitative determination of analyte concentration.
In either nephelometric or turbidimetric immunoassay, the photometer can be used to measure the extent of change of an optical property of the liquid medium after the immunoprecipitation reaction has essentially run to completion (i.e. an end point determination). Alternately, the photometer can be used to detect the rate of change of an optical property of the liquid medium at a particular time after commencement of the immunoprecipitation reaction (i.e. a rate determination). Immunoassay by known end-point or known rate photometric methods presents problems and inefficiencies, particularly where an objective is to detect and quantify drug and protein levels in a large number of physiological fluid samples in the shortest possible time.
With regard to photometric end point immunoassays, these problems include the inherent slowness of end point methods. Thus, with end point methods, the immunoprecipitation reaction is allowed to go to or near reaction completion, before a photometric reading of light scatter or increased liquid medium turbidity is made. Hence, analyte detection is delayed until at least about five minutes or more after the immunoprecipitation reaction has begun. Additionally, the throughput or analysis rate per unit of time where multiple samples are to be analyzed is low. Such delays force concomitant delays in diagnostic and therapeutic decision-making.
Additionally, photometric end point immunoassay methods generally require at least some sample manipulation prior to analysis. Typically, dilution of an analyte-containing physiological fluid sample is carried out by, for example, adding an aliquot of the physiological fluid to saline. A dilution step is required because besides analyte, the physiological fluid usually also contains many other substances such as proteins, steroids, hormones, drugs and a variety of metabolites, that can cause nonspecific immunoprecipitation reactions or otherwise affect optical properties of the liquid medium. Thus, a very high level of interfering or background light scatter and light absorbance occurs when an aliquot from an neat (i.e. undiluted) sample of analyte-containing physiological fluid is used. Hence, end point determination of analyte with an undiluted physiological fluid sample is very difficult or impossible.
Any sample manipulation, including sample dilution, is undesirable because it delays sample analysis and inherently carries the risk of the sample becoming contaminated or of otherwise interfering with an accurate analyte detection and quantification. Furthermore, sample manipulation, by increasing worker human contact time with the sample, increases the risk of disease transmission from sample to the sample handler.
Photometric rate immunoassay methods also have deficiencies. Maximum rate nephelometry and peak rate turbidimetry detect analytes by measuring the maximum rate of a changing optical characteristic of the liquid medium in which the immunoprecipitation reaction takes place. A first deficiency exists because in these known methods, the photometrically detected maximum or peak rate typically does not occur until at least about a minute or more after the beginning of the photometrically detectable immunoprecipitation reaction. Hence, as with end point immunoassay methods, the known rate methods are slow and prevent achievement of a high sample throughput per unit time.
A second problem with photometric rate immunoassay methods is due to the nature of the rate signals detected. The existing rate immunoassay methods typically do not detect a substantially constant or linear-with-time photometric rate signal. A nonlinear-with-time rate signal can require use of time consuming and complicated mathematical formulae to fit the nonlinear rate curve derived from the signal to a linear approximation. Rapid analyte detection and high sample throughput are thereby hindered.
A third problem with known photometric rate immunoassay methods arises from the variability of the time at which the peak or maximum rate signal occurs. Depending on factors such as analyte concentration, Specific binding partner concentration, and nature of the analyte and the specific binding partner, the maximum or peak rate signal can occur at widely varying times after initiation of a photometrically detectable immunoprecipitation reaction. Thus, when dealing with one or more unknowns, such as analyte concentration, it can not be determined beforehand when the analytically useful maximum or peak rate signal will occur. Hence, the photometer used is either set to detect signal at a particular time or times, in hopes of catching the right signal, or the immunoprecipitation reaction is tracked continuously until the desired signal occurs. This uncertainty regarding when the maximum or peak rate signal will occur leads to loss of time, inefficiency in analysis, and additional expense.
Thus, there is a need for a photometric immunoassay method that permits: (1) rapid analyte detection; (2) analyte detection in both undiluted (neat) and diluted samples; (3) accurate analyte quantification; (4) high sample throughput per unit time; and (5) low per-sample analyzed cost.